tbuckley/cabot-search huggingface dataset:
Abstract P1057: Implementing A Novel FRET-Based Myofilament Activation Biosensor To Interrogate Dynamic Cardiac Sarcomere Performance With Small Molecule And Genetic Manipulations During A Physiological Twitch
Authors: Ashley A Martin, Brian R Thompson, Joseph M Metzger
Journal: Circulation Research (2022)

The sarcomere is the functional unit of cardiac muscle contraction. Understanding the precise mechanisms regulating this contraction is key to advancing insights into cardiomyopathies, which affect as many as 1 in 500 adults. Previous work has identified many key sarcomeric components and modulators necessary for the activation of contraction through inter-myofilament signaling. Although these studies have provided valuable information, the majority of this work has been done using in vitro protein recombination or permeabilized muscle. While informative, these systems lack significant physiological features of muscle, including load and intact excitation-contraction coupling mechanisms. Therefore, a large gap exists in the ability to monitor the time-sensitive stepwise physiological mechanism of muscle sarcomere activation in an intact system in real time. In order to further advance the study of sarcomere activation in both physiological and disease states, a cardiac specific myofilament-targeted FRET based biosensor has been designed and validated. The sarcomere activation biosensor reports myofilament dynamics during physiological twitch contractions in live cardiac muscle in real time in the form of simultaneous force and fluorescence measurements. This allows FRET-detection of the global conformational changes in TnC, a nexus point able to integrate many sarcomere-activating ligands, to be observed. From these changes in fluorescence the ensemble of events which lead to sarcomere activation and muscle contraction can be tracked. Data show regulatory ligands of myofilament activation in real time during a physiological twitch contraction. Currently, the biosensor is being utilized to investigate changes in sarcomere activation driven through the use of small molecules, including myosin inhibitor Mavacamten and TnC-TnI inhibitor W7, and those seen in MyBP-C knockout mice. The results of these investigations will be discussed.
Abstract P3096: Cardiac-specific Hadc3 Loss Induces Reorganization Of Peripheral Heterochromatin And Promotes Diet-induced Heart Failure
Authors: Anna Kiseleva, Cheryl L Smith, Jonathan A Epstein
Journal: Circulation Research (2022)

The nuclear periphery, including the nuclear lamina, serves as an important three-dimensional organizer of the genome. Together, with a subset of tethering proteins, nuclear lamina serves as a platform to anchor transcriptionally silent heterochromatin to the nuclear periphery. Peripheral heterochromatin, marked by the H3K9me2 histone modification, interacts with lamina proteins and is organized into lamina-associated domains (LADs). Our previous studies demonstrated that histone deacetylase 3 (HDAC3) is a scaffold for a nuclear lamina tethering complex and plays an important, non-enzymatic role in establishing cell identity in murine cardiac progenitor cells by reorganizing the LADs and releasing cardiac lineage-specific genes from the nuclear periphery during differentiation. This reorganization is critical as it makes specific genomic regions that are released from the nuclear periphery more accessible for transcriptional activation. Surprisingly, Hdac3 deficiency in murine cardiomyocytes did not lead to any observable phenotype in adult animals. However, when the mice were maintained on a high-fat diet, this resulted in a severe disruption of fat metabolism and lead to heart failure within months. In the present study, we seek to determine if Hdac3 deletion is sufficient to re-organize LADs, release the genes normally found there, and make them more accessible for transcriptional activation as the mechanism leading to cardiac dysfunction. Since H3K9me2 marks peripherally localized genomic regions, we performed H3K9me2 CUT&amp;RUN assay on cardiomyocytes derived from WT and Hdac3 KO mice, fed with normal and high-fat diet, to compare the positioning of chromatin genome-wide. From these data, we identified a list of genes and enhancers found in those regions that vary significantly in H3K9me2 enrichment depending on genotype and condition. We then performed Gene Ontology (GO) Analysis. A significant finding is that numerous enhancers with relatively reduced H3K9me2 in Hdac3 KO cardiomyocytes are regulators of neuronal genes. Loss of H3K9me2 is generally associated with increased gene transcription. We predict that disruption of the peripheral heterochromatin organization upon Hdac3 deletion may result in loss of identity in cardiomyocytes by activating non-cardiomyocyte program-specific genes, such as those of neuronal cell fate, which are normally found at the nuclear periphery and silent in cardiac cells. The results of this work will help to gain insight into the function of nuclear lamina scaffolding proteins, and open new avenues to design therapies to replace lost scaffolding proteins in patients predisposed to heart disease.
Abstract P2007: Bmal1 Drives Postnatal Cardiac Hypertrophy Via Circadian Chromatin Remodeling Of The Pro-hypertrophic Gene <i>Sik1</i>
Authors: Adrian Arrieta, Anna Reese, David Wong, Douglas J Chapski, Manuel Rosa-Garrido, Ashley Zhu, Thomas M Vondriska
Journal: Circulation Research (2022)

Background: During neonatal cardiac development, myocytes integrate cues such as changes in blood pressure and circulating hormones to execute a temporal genetic response that drives physiological cardiac hypertrophy. Chromatin remodeling precedes the adult heart’s response to growth stimuli, yet the temporal nature of neonatal chromatin remodeling is unclear. In other tissues, genes regulated by the master circadian rhythm factor BMAL1 display circadian oscillations in histone acetylation and nucleosome and RNAP II occupancy. We hypothesized that BMAL1 regulates temporal chromatin remodeling and expression of genes critical for neonatal cardiac hypertrophy. Methods: BMAL1 chromatin immunoprecipitation sequencing (ChIP-Seq) and assay for transposase-accessible chromatin and sequencing (ATAC-Seq) data from murine hearts were analyzed to identify genes at which BMAL1 may drive chromatin remodeling. To interrogate myocyte time-of-day dependent response to growth stimuli, neonatal rat ventricular myocytes (NRVM) were subjected to serum shock synchronization to drive oscillatory BMAL1 expression, followed by treatment with the growth stimulus phenylephrine (PE) at the peak and trough of BMAL1 expression. BMAL1 was knocked down via siRNA and hypertrophy was assessed by measurement of cell size and of RT-PCR of fetal genes (Nppa and Nppb). Results: BMAL1 ChIP-Seq data revealed myocyte-specific BMAL1 localization to regions exhibiting an open chromatin signature based on ATAC-seq, including the pro-hypertrophic gene salt-inducible kinase 1 ( Sik1 ). Serum shock induced oscillations of BMAL1 and histone H3.3: administration of PE at the peak of BMAL1 expression induced a 30% increase in cell size and 2-fold increase in fetal gene expression. PE given at the trough of BMAL1 expression affected neither cell size nor fetal genes, demonstrating a critical role for the circadian clock in myocyte growth. BMAL1 knockdown: decreased expression of its known target and partner clock gene, Per2 , as well as expression of Sik1, Nppa , and Nppb ; decreased myocyte size by 30%; and increased expression of histone H3.3. Conclusions: These data indicate that BMAL1 temporally regulates myocyte growth by modulating histone stoichiometry and expression of Sik1 .
Airway goblet cells: responsive and adaptable front-line defenders
Authors: DF Rogers
Journal: European Respiratory Journal (1994)

Goblet cells are situated in the epithelium of the conducting airways, often with their apical surfaces protruding into the lumen, a location which fits them for a rapid response to inhaled airway insults. Together with the submucosal glands, goblet cells secrete high molecular weight mucus glycoproteins (mucins), which confer upon the airway surface fluid the requisite biochemical and biophysical properties which determine the efficiency of entrapment and transportation of inhaled irritants, particles and micro-organisms. The diversity of glycosylation of airway mucins may be important in facilitating adherence of micro-organisms to mucus prior to mucociliary clearance. Other secretory products, including lipids and "small" glycoproteins, may also be produced by goblet cells. It is possible that goblet cells have the potential to produce markedly more mucus than do the glands. Mucins are tightly packed in the intracellular granules of the goblet cell. The morphology of these granules varies with fixation technique, and release of mucins may be via a combination of merocrine and apocrine secretion. Discharge of mucus is accomplished remarkably rapidly (tens of milliseconds) and vast quantities of mucus are released (size expansions from the granule of many hundredfold). Depending upon species and preparation, goblet cells discharge mucus in response to a wide variety of stimuli, including proteinases, irritant gases, inflammatory mediators, reactive oxygen species, nerve activation and changes in the biophysical environment. Under normal conditions, goblet cell proliferation and differentiation, particularly to ciliated cells, contributes to maintenance of the airway epithelial cell population. In addition to participating in acute airway defence, goblet cells increase in number in response to chronic airway insult, with a resultant increase in output of mucus. The increase in number of cells is via hyperplastic and metaplastic mechanisms. Early triggers for the development of a hypersecretory epithelium include excessive discharge of mucus and increased expression of airway mucin messenger ribonucleic acid (mRNA). Cessation of chronic airway stress rapidly reverses the increased number of goblet cells. Irritant-induced increases in number of goblet cells can be inhibited by a variety of drugs with anti-inflammatory and mucoregulatory properties, and the reversal to normal numbers after cessation of the irritation is speeded by these drugs. The ability of goblet cells to be progenitors of ciliated cells, to rapidly produce vast quantities of mucus in response to acute airway insult, and to change in number according to variations in chronic insult indicates that these cells are vitally important responsive and adaptable front-line defenders of the airways.
Abstract P2053: Role Of Podoplanin Positive Cells Exosomes In Cardiac Inflammation And Amyloidosis
Authors: Maria Cimini, Carolina Gonzalez, Cagla Tukel, Mary Barbe, Anna Maria Lucchese, Chunlin Wang, MAY TRUONGCAO, Grace Huang, Andrea Elia, Vandana Mallaredy, Cindy Benedict, Raj Kishore
Journal: Circulation Research (2022)

The extra-cellular-matrix (ECM) composition of scar tissue after myocardial infarction (MI)has been largely investigated. Although fibronectin and collagen are favorable for newmyocyte formation, other components that may increase the scar stiffness and reducethe remodeling of the ischemic area, remain to be identified. Our preliminary studiesidentified primary Serum Amyloid A3 (SAA3) extracellular accumulation that maycontribute to the chronic alteration of the ischemic myocardium’s scar. Specifichistological staining such as thioflavin and Congo red, showed amyloid deposition inmouse hearts 1 month after MI; furthermore, immunohistochemistry for SAA3 detectedthe deposition of the misfolded protein alongside fibronectin and collagen. Serum amyloidamyloidosis (AA) is characterized by deposition of hepatic misfolded protein and SAA3 isthe only amyloid protein that is released locally after inflammation, mostly bymesenchymal progenitor cells. We have reported earlier that two days after MI, a cohortof mesenchymal cells begin to de novo express Podoplanin (PDPN), a plateletaggregation-inducing type I transmembrane glycoprotein, as a signal of activation.PDPN+ cells, in addition to cytokines, release extracellular vesicles including exosomes(Exo) as major paracrine entities driving intercellular communications in homeostasis anddisease. Exo derived from activated PDPN+ cells isolated from MI hearts highly expressSAA3 and injection of activated PDPN+ cell Exo in uninjured healthy mouse hearts leadsto recruitment of immune cells, an extended epicardial fibrosis and amyloidosis with asubsequent impairment in the contractility and increase of the end systolic volumes anddiameters. SAA3 binds Toll-like receptors, and in vitro treatment of bone marrow derivedmonocytes either with PDPN+ cells derived Exo or recombinant SAA3, activated themtowards pro-inflammatory phenotype on contrary these stimuli failed to activate TLR2knocked out monocytes showing an impairment in the expression of major cytokine,chemokine and pro inflammatory markers. Thus, PDPN+ cells in the ischemic heartrelease SAA3 through Exo prolonging inflammation and macrophage recruitment viaTLR2 and contribute to amyloidosis after MI.
Abstract P1006: Cross-lineage Potential Of Ascl1 Uncovered By Comparing Diverse Reprogramming Regulatomes
Authors: Haofei Wang, Benjamin Keepers, Jiandong Liu, Li Qian
Journal: Circulation Research (2022)

Direct reprogramming has revolutionized the fields of stem cell biology and regenerative medicine, making it possible to interconvert somatic cell fates without intermediary pluripotency. Although studies have identified different transcription factor (TF) cocktails that can reprogram fibroblasts into different cell types, the common mechanisms governing how reprogramming cells undergo transcriptome and epigenome remodeling (i.e., regulatome remodeling) have not been investigated. Here, by characterizing early changes in the regulatome of three different types of direct reprogramming - induced neurons, induced hepatocytes, and induced cardiomyocytes - we identify lineage-specific features as well as common regulatory transcription factors that act in a context-dependent manner for each type of reprogramming. Of particular interest, we discover that the neuronal factor Ascl1 possesses cross-lineage potential; together with Mef2c, it navigates reprogramming fibroblasts through a targeted and potent iCM trajectory. Single-cell multi-omics reveal that A+M reprogramming terminates in a more mature iCM phenotype than MGT. Finally, through ChIP-seq and RNA-seq, we find that Mef2c drives the shift in Ascl1 binding away from neuronal genes towards cardiac genes, guiding their co-operative epigenetic and transcription activities. Given the relatively short DNA coding sequence of Ascl1 (696bp) and Mef2c (1299bp), our novel A+M reprogramming cocktail has the potential to be delivered within a single AAV vector. Such a strategy is not feasible with classic cocktail MGT (4182bp). Together, our study demonstrates the existence of common regulators of different direct reprogramming processes, argues against the premise that TFs possess only lineage-specific capabilities for altering cell fate - the basic premise used to develop direct reprogramming approaches and discover a novel cardiac reprogramming cocktail showing great potential for future clinical application.
Abstract GS113: Inflammasome Primed Neutrophils Regulate Myocardial Infarction Induced Myelopoiesis
Authors: Gopalkrishna Sreejit, Sunil Nooti, Robert Jaggers, Jillian Johnson, Jianjie Ma, Andrew Murphy, Prabhakara R Nagareddy
Journal: Circulation Research (2022)

Background: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in-part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1β signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 inflammasome and associated signaling components in neutrophils, the serum levels of IL-1β, the effector molecule in granulopoiesis was not impacted by MI suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. Methods: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the LAD artery, and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in non-infarcted parabionts. Finally, utilizing multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1β secretion and granulopoiesis. Cardiac function was assessed by echocardiography. Results: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the non-infarcted parabionts. Prior priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed neutrophils secrete IL-1β through formation of gasdermin D pores and, promote granulopoiesis. Pharmacological and/ or genetic strategies aimed at inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and, improved cardiac function. Conclusions: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (e.g., IL-1β) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release
Abstract P1061: Crosstalk Between Acetylation And Methylglyoxal Modifications On Myofilament Proteins Affects Sarcomere Function
Authors: Christine Delligatti, Maria Papadaki, Thomas Martin, Jonathan A Kirk
Journal: Circulation Research (2022)

Diabetes doubles the risk of developing heart failure, independent of coronary artery disease and hypertension. The mechanisms connecting these two diseases are poorly understood, although we have previously shown one connection is likely due to methylglyoxal (MGO), a highly reactive glycolysis byproduct that can irreversibly modify lysine (K) and arginine (R) residues in a process called glycation. We found MGO glycation of the myofilament decreased calcium sensitivity and maximum calcium activated force in diabetes. Here we hypothesized MGO may further impact function by competing for lysine residues that would otherwise be targets for acetylation, which has been shown to impact cardiac function. To assess this hypothesis, non-failing human left ventricular (LV) myocardial tissue was exposed to MGO (100 μM) or vehicle and then the treated with acetic anhydride in acetonitrile (300 μM, Ac 2 O-ACN) overnight. Ac 2 O-ACN increased myofilament protein acetylation in both groups, but this effect was greatly reduced in samples pre-treated with MGO, suggesting these two PTMs do compete for a subset of lysine residues. To discover the specific sites of this acetylation-glycation crosstalk, we compared mass spectrometry data sets from diabetic humans, MGO treated mouse myocytes, and acetylated myocytes, which revealed sites of crosstalk on actin, myosin, and myosin light chains - proteins essential for normal contractile function. Importantly, these glycation-acetylation crosstalk sites were found in human samples and the data suggest the balance of these may change in disease states. To explore the functional consequence(s) of this crosstalk, we incubated human LV isolated skinned myocytes with Ac 2 O-ACN (300 μM, overnight), which enhanced function by significantly increasing calcium sensitivity. However, pre-treatment with MGO (100 μM) completely blocked this functional effect of acetylation. Together, these data demonstrate that glycation and acetylation compete for certain myofilament lysine residues, and that glycation can block the beneficial functional effects of acetylation. These results provide insight into the molecular mechanisms behind diabetic cardiomyopathy and the utility of targeting acetylation clinically.
Abstract P3027: Intermittent Fasting Improves High Fat Diet Mediated Heart Failure With Preserved Ejection Fraction
Authors: Guodong Pan, Bipradas Roy, Ramandeep Rattan, Suresh S Palaniyandi
Journal: Circulation Research (2022)

Intermittent fasting (IF) has improved metabolic aberrations in metabolic diseases, however, the mechanism is unclear. Our lab focuses on 4-hydroxy-2-nonenal (4HNE), an oxidative stress byproduct which accumulates in metabolic diseases, in cardiac damage and activity of aldehyde dehydrogenase 2 (ALDH2), a 4HNE detoxifying enzyme, which was implicated in cardioprotection. We reported that the ALDH2*2 knock-in mutant mice (AL) with low intrinsic ALDH2 activity that were fed with high-fat diet (HFD) exhibited severe heart failure with preserved ejection fraction (HFpEF) compared to HFD fed control mice (C57). We hypothesized that IF can contribute to the improvement of HFpEF even in AL mice by reducing 4HNE. C57 and AL mice that were fed HFD and L-NAME (nitric oxide inhibitor) in drinking water were grouped as C57-free and AL-free meaning taking food freely or C57-IF and AL-IF meaning daily 16 hours fasting with 8-hr free access to food. After 2-months of HFD feeding, the body weights of C57-IF (35.5±3.8g) and AL-IF (36.7±3.4g) groups were significantly lower than C57-free (43.5±4.2g, p&lt;0.05 vs C57-IF) and AL-free (45.8±5.6g, p&lt;0.05 vs AL-IF) groups. The 24-hour food intakes of C57-IF (8.6±0.9 KCal) and AL-IF (8.8±1.1 KCal) were significantly decreased, compared to C57-free (12.3±1.7 KCal, p&lt;0.05 vs C57-IF) and AL-free (13.3±1.5 KCal, p&lt;0.05 vs AL-IF) groups. The respiratory exchange ratio (RER) of AL-free group (0.62±0.12) was significantly lower than three other groups (p&lt;0.05 vs C57-free, C57-IF and AL-IF, respectively). The E/A ratio (E/A), a diastolic parameter, was decreased in C57-free (1.14±0.21) and AL-free (0.89±0.17) groups, not in C57-IF (2.15±0.31, p&lt;0.05 vs C57-free) and AL-IF (1.97±0.25, p&lt;0.05 vs AL-free) groups. The E/A in AL-free group was also lower than C57-free group (p&lt;0.05). Another diastolic parameter, isovolumic relaxation time (IVRT) was also prolonged in C57-free (27.3±3.3 ms) and AL-free (29.4±3.7 ms) mice, not in C57-IF (13.3±1.8 ms, p&lt;0.05 vs C57-free) and AL-IF (15.2±2.1 ms, p&lt;0.5 vs AL-free) mice. In conclusion, IF decreased food intake and obesity, and improved metabolism and heart function in both C57 and ALDH2*2 mice with HFpEF, and also diminished the differences in RER and E/A between them via lowering the 4HNE adducts.